PB0874 - The ins and outs of platelet contractility during thrombus formation

Background: Non-muscle myosin IIA plays important roles in proplatelet formation by megakaryocytes and in thrombus formation by platelets, as illustrated by macrothrombocytopenia and defective clot contraction in MYH9-related disorders (MYH9-RD), respectively. Recent work showed that reduced traction forces of MYH9-RD platelets are causative for bleeding (doi: 10.1126/sciadv.abn2627), and that platelets adjust their contractility in response to adhesion proteins found in the thrombus core or in the basement membrane (doi: 10.1126/sciadv.abj8331).

Aims: To further elucidate the contribution of myosin IIA activity to diverse platelet functions and its regulation by the thrombus microenvironment.

Methods: Washed platelets were treated with pharmacological inhibitors and/or platelet agonists and analysed for traction forces on fibrinogen-coated micropost arrays, initial interactions with VWF under flow, spreading on fibrinogen, ultrastructure of the actin cytoskeleton and focal adhesions, aggregation in solution, ATP release, and the phosphorylation of kinases involved in the regulation of myosin activity.

Results: Mild myosin inhibition by low-dose blebbistatin eliminated highly contractile platelets yet maintained basic adhesive, aggregatory and secretory platelet functions. Severe contractility loss impaired adhesion receptor-cytoskeleton coupling and, unexpectedly, dense granule secretion and platelet aggregation downstream of GPVI. Thrombin or the combination of PAR1 and PAR4 activating peptides increased platelet traction forces more than stimulation of PAR1 or PAR4 alone or agonists for GPVI, P2Y receptors. Prolonged actomyosin contractility was maintained by an elevated myosin light chain expression and the activity of regulatory kinases. Inhibition of P2Y receptors or the COX pathway, as in (dual) antiplatelet therapy, diminished platelet traction forces, whereas novel GPVI antagonists did not significantly impair contractility.

Conclusion(s): The picture emerges that adhesion proteins and soluble agonists of the core-shell thrombus structure locally orchestrate the development of platelet contractile forces. Our findings support the hypothesis that highly contractile platelets are required for haemostasis. Further studies are needed to determine how myosin IIa activity contributes to GPVI signalling.